Lyophilized emulsion containing an active substance

ABSTRACT

The invention concerns a freeze-dried emulsion composition containing an active substance and which can be reconstituted using water to give the original emulsion, plus a method for producing the freeze-dried composition. The reconstituted emulsion is suitable for parenteral use.

This is a Continuation of application Ser. No. 08/491,862, filed Jun.23, 1995, now U.S. Pat. No. 5,612,058, which application(s) areincorporated herein by reference.

DESCRIPTION

The present invention relates to a lyophilized emulsion which can beredispersed with water to give the original emulsion.

In particular, the invention relates to a fat emulsion which contains anactive substance and whose external aqueous phase has been removed byfreeze-drying and which can be redispersed by addition of waterspontaneously to give the original emulsion with a particle sizedistribution corresponding to the initial formulation.

Emulsions are disperse systems composed of two mutually immiscibleliquids, one of which, the internal, disperse phase, is finely dispersedin the other, the external, continuous phase.

Fat emulsions are emulsion systems in which the internal, disperse phaseconsists of very fine fat particles which are homogeneously dispersed inthe external phase which is composed of water. Emulsion formulations ofthis type are preferably used parenterally and are particularly used forintravenous nutrition of patients unable to take food by mouth.

Fat emulsions which can be administered intravenously make high demandson the tolerability of their ingredients and the particle size of thefat particles. Preferably used as fat component are oils with a highcontent of unsaturated fatty acids such as soya bean, safflower andcottonseed oils, as emulsifiers are lecithins such as egg, soya andcerebral lecithins, as well as antioxidants such as tocopherol acetateand other auxiliary substances.

The fat particles should, in order to avoid changes in blood pressureand the risk of embolism, not exceed an average particle size of 1 μm.

The emulsion is normally prepared by preemulsifying the heated oil andaqueous phases with a mixer, followed by microfine emulsification usinga high-pressure homogenizer and subsequent sterilization withsuperheated steam.

The "Handbook on Injectable Drugs" (American Society of HospitalPharmacists, pages 237-244 (1986), Lawrence A. Trissel) describes somecommercially available formulations. They contain soya bean oil orsafflower oil, egg lecithin, glycerol and water and have averageparticle sizes of ≦0.5 μm.

Fat emulsions are also repeatedly used as vehicle systems for lipophilicmedicinal substances to be administered parenterally. The aim in thiscase is to increase the therapeutic efficacy and safety of medicinalsubstances by controlled release from emulsion systems.

In accordance with their solubility properties, lipophilic activesubstances in emulsions are partly or completely incorporated in the fatparticles. This means that their pharmacokinetic behaviour is cruciallydetermined by the pharmacokinetic behaviour of the vehicle formulationsfrom which the active substance is first released. Delayed releaseavoids high local concentrations of active substance, reducesdegradation and thus increases the duration of action.

Emulsion systems of this type are particularly advantageous forprostaglandins, especially prostaglandin E₁ (PGE₁). PGE₁ is a highlyactive tissue hormone which is successfully used, for example, for thetreatment of arterial occlusive disease. Used for this purpose is a PGE₁-α-cyclodextrin complex which, dissolved in physiological salinesolution, is infused parenterally, preferably intraarterially, as closeas possible to the body region to be treated. However, high pressureconditions and small dilution effects during the intraarterial infusionmake high demands on the equipment and the training of the treatingphysician. Although intravenous infusion is simpler to perform bycomparison, even in this case infusion is possible only slowly and inrelatively high dilution because of the local irritant effect of PGE₁.Overall, the extended residence time of the active substance in thevascular system before reaching the target site and, in particular, theadditional passage through the pulmonary circulation leads to increaseddegradation of active substance. Both intraarterial and intravenousinfusions make high demands on the equipment and careful adjustment ofthe infusion rate and are therefore usually performed in hospital andnot by the established physician, which impedes wide use of the valuableactive substance in the therapy of arterial occlusive disease.

These problems can be avoided by incorporating PGE₁ in a fat emulsion.Delayed release of active substance avoids high local concentrations,reduces degradation of active substance and increases the duration ofaction so that formulations of this type are also suitable forintravenous bolus injection.

The process for preparing such fat emulsions containing activesubstances substantially corresponds to the above-mentioned preparationof a fat emulsion, with the difference that the active substance to beincorporated is dissolved in the oil phase before carrying out thepreemulsification. Although PGE₁ -containing fat emulsions of this typeare suitable for solving the disadvantages described for theconventional use of PGE₁, they have low storage stability owing tohydrolytic degradation of the active substance, which impedes theirgeneral utilizability.

One possibility for stabilizing the active substance within the emulsioncomprises removal of the substances destabilizing the active substance.One example of this is given in U.S. Pat. No. 4,684,633 which describesthe stabilization of active substances brought about by usingphosphatidylethanolamine-free egg lecithin in an emulsion compositioncontaining prostaglandin, soya oil, egg lecithin, glycerol and water.However, stabilization of active substances is shown only for thecondition of brief sterilization at 125° C. for 2.2 min. There are nodata on the stability on long-term storage. The formulation alsocontains water so that degradation of active substance as a result ofhydrolysis cannot in principle be ruled out. A fundamental disadvantageis that the stabilization occurs only to the stated active substancesand is not generally applicable.

Besides stabilization of the active substance in a fat emulsion readyfor administration, fat emulsions with intact active substance can alsobe used by being prepared only immediately before use. One example ofthis is given by EP 0 331 755. It describes a kit consisting of aconventional fat emulsion and either an active substance solution inwater, liquid polyalkylene glycols, liquid alkylethanolamines or liquidalcohols containing a plurality of hydroxyl groups, or an activesubstance composition consisting of active substance, saccharides and/oramino acids, which are combined and vigorously mixed immediately beforeuse. Vigorous mixing is absolutely necessary to make it possible todisperse the active substance in the fat emulsion. Thus, for example,the active substance emulsion described in Example 5 is prepared bymixing for 2-3 minutes. However, long mixing times are disadvantageouson use.

An example relating to prostaglandins which is mentioned in EP 0 331755, Example 3, describes an active substance composition consisting ofa prostaglandin and triethanolamine. However, triethanolamine is notwithout objections physiologically so that its use in pharmaceuticalformulations, especially in injectable products, should be avoided wherepossible.

There has thus been a continuing need for an emulsion formulation whichavoids PGE₁ degradation as a consequence of hydrolysis, isphysiologically unobjectionable and is simple to handle. A formulationof this type ought to preclude in principle hydrolytic degradation ofactive substance on storage due to absence of water, be easilyredispersible by addition of water, and ensure dispersion of the activesubstance in the fat phase of the emulsion from the outset.

A formulation of this type is to be prepared by lyophilization of a fatemulsion containing active substance, which as anhydrous formulationleads to the expectation of storage stability and can be reconstitutedwith water to give the original formulation before administration.However, investigations show that lyophilization of emulsions lead tocoalescence of the fat particles and thus to enlargement of theparticles thereof or even to their complete destruction. Variousresearch groups have repeatedly attempted to prevent this coalescence ofemulsions as a consequence of lyophilization by adding cryoprotectionagents. However, their results show that even this measure is unable toprevent an increase in the average particle diameter Lladser, M. et al.:The use of supports in the lyophilization of oil-in-water emulsions, J.Pharm. Pharmacol 20, 450-455 (1968); Rambhan, D. et al.: StabilityStudies on Lyophilized O/W Emulsions, Indian J. Pharm. 39, 52-55(1977)!. Bensouda et al. further show that the increase in the averageparticle diameter as a consequence of lyophilization increases withdecreasing size of the emulsion particles Bensouda, Y. et al.:Freeze-drying of Emulsions--Influence of Congealing on GranulometryResearch of a Cryoprotection Agent, Pharm. Acta Helv. 64, 40-44 (1989)!.However, emulsions for intravenous administration should, in order toavoid embolism, have an average particle size less than or equal to 1μm. Prevention of an increase in particle size as a consequence oflyophilization therefore appears to be particularly difficult, but isindispensible for safety reasons.

Lyophilized reconstitutable emulsions which are said also to be suitablefor intravenous use are described in the documents JP 60239417 and ZA 8604 032. JP 60239417 describes the preparation of lyophilized emulsionsystems with the addition of cryoprotection agents such as saccharidesand sugar alcohols and water-soluble polymers such aspolyvinylpyrrolidone, gelatin and hydroxypropylcellulose. However, it isevident from the examples that the particle diameters of the redispersedemulsions are all higher than in the initial emulsion. ZA 86 04 032discloses lyophilized emulsion compositions and a method for theirpreparation. After preparation of a conventional fat emulsion by knownprocesses and addition of a bulking sugar, the emulsion is sprayed intoa boiling liquid with a boiling point below -20° C. and subsequentlylyophilized. The examples show, however, that even in this case theaverage particle diameter of the fat particles after reconstitution withwater is higher than in the initial emulsion.

Lyophilized emulsions which have, after reconstitution with water, aparticle dispersion corresponding to the initial formulation are notpresent in the prior art.

Surprisingly, an anhydrous emulsion composition which contains activesubstance and which contains at least one cryoprotection agent/bulkingagent and can be redispersed by addition of water to give the original,water-containing, active-substance containing emulsion withcorresponding particle size distribution has now been found. An emulsioncomposition having no coherent external aqueous phase is termedanhydrous. A composition of this type avoids hydrolytic degradation ofthe active substance(s) and shows high storage stability.

The emulsion composition advantageously contains hydrophilic emulsifiersand acetylated monoglycerides. Hydrophilic emulsifiers are surfactantswhose emulsifying behaviour is crucially determined by their hydrophilicgroups and preferably form fat-in-water emulsions.

The emulsion composition preferably contains ethoxylated triglyceridesor polyoxyethylene hydroxy fatty acid esters and acetylatedmonoglycerides with unsaturated double bonds.

The emulsion composition particularly contains glycerol polyethyleneglycol ricinoleate (macrogol glycerol ricinoleate) or polyoxyethylene660 12-hydroxystearate (macrogol 660 12-hydroxystearate) anddiacetylated monoglycerides or a mixture of diacetylated and partiallyacetylated monoglycerides.

Particularly advantageous in this case are acetylated monoglycerideswhich contain 2% by weight to 40% by weight, preferably 20% by weight,of partially acetylated monoglycerides.

According to an expedient embodiment of the present invention, thehydrophilic emulsifiers and the acetylated monoglycerides are present ina ratio of 1:10 to 2:1 by weight, preferably in a ratio of 2:3 byweight.

According to a particularly preferred embodiment of the anhydrousemulsion composition according to the invention, the latter contains atleast one active substance from the active substance group ofprostaglandins, especially PGE₁.

Another advantageous embodiment of the emulsion composition according tothe invention contains as cryo-protection agent/bulking agentphysiologically tolerated mono-, di- or oligosaccharides, especiallylactose or sugar alcohols such as sorbitol and/or mannitol.

According to another preferred embodiment of the emulsion compositionaccording to the invention, the latter contains at least one customaryantioxidant, advantageously from the group of tocopherols such as α-,β-, γ- or δ-tocopherol, preferably α-tocopherol, and the physiologicallytolerated salts thereof, such as phosphates, succinates and acetatesand/or physiologically tolerated buffer salts.

According to another preferred embodiment of the emulsion compositionaccording to the invention, its internal disperse phase has, afterreconstitution with water, average particle diameters of 0.1 μm to 5 μm,preferably 0.2 μm to 1.0 μm.

Another particularly preferred embodiment of the anhydrous emulsioncomposition according to the invention contains cryoprotectionagents/bulking agents and can be redispersed by addition of water togive the original water-containing emulsion with substantially or nearlyidentical particle size distribution.

The emulsion composition according to the invention can be prepared byremoving the aqueous phase by lyophilization from an emulsion which hasbeen prepared by the processes and technologies customary in theproduction of pharmaceuticals.

The invention therefore also relates to a process for the preparation ofthe active-substance containing anhydrous emulsion composition,according to the invention, which is characterized in that anactive-substance containing emulsion is prepared in a conventional wayand its external aqueous phase is subsequently removed by freeze-drying.

It is possible to incorporate in the emulsion composition according tothe invention hydrophilic active substances by dissolving in the aqueousphase, and lipophilic active substances by dissolving in the phasecontaining emulsifier and fat. Alternatively, the active substance canalso be added immediately before the lyophilization is carried out,which is particularly advantageous for active substances which aresensitive to hydrolysis and/or unstable to heat. This means that theprocess for preparing the emulsion composition containing an activesubstance according to the invention can be adapted in an advantageousmanner to suit the physicochemical properties of the active substances.Therefore, in an expedient embodiment of the process according to theinvention, at least one active substance is dissolved either in theaqueous phase or in the phase containing emulsifier and fat beforeemulsification, or at least one active substance is added to theemulsion before it is freeze-dried. Care should be taken in this casethat the active substance dispersion within the disperse system isensured before lyophilization, which can easily be checked by customarymethods such as equilibrium dialysis, differential dialysis andultrafiltration.

PREPARATION EXAMPLE 1

emulsion preparation

3.42 g of citric acid monohydrate, 1.57 g of trisodium citrate dihydrateand 60.0 g of lactose were dissolved by heating in 475 g of water forinjections. Subsequently, 12.0 g of polyoxyethylene 66012-hydroxystearate and 18.0 g of diacetylated monoglycerides with ahydroxyl number of 25 were dissolved in 30.0 g of absolute ethanol byheating to about 30° C. under an inert atmosphere (nitrogen).

The aqueous phase was transferred into a suitable presterilized reactionvessel (IKA LR-A 1000 laboratory reactor, IKA-Werke, Jahnke & KunkelGmbH, Staufen, Germany) with temperature-control device, stirrer tooland toothed rim disperser (Ultraturrax, IKA-Werke, Jahnke & Kunkel GmbH,Staufen, Germany) and heated to 80° C. while stirring under a vacuum of<1 mbar. While maintaining the vacuum and the stirring, the ethanolicemulsifier lipid phase was slowly injected through a cannula directlyinto the aqueous phase with simultaneous vigorous homogenization usingthe Ultraturrax. The mixture was subsequently cooled, with continuousstirring and maintenance of the vacuum, to room temperature whilevigorous homogenization was carried out using the toothed rim dispersingrod for about 1 min in several periods. The cooled emulsion wastransferred into a presterilized bottle and stored in a refrigeratoruntil processed further.

To incorporate the active substance, 163.9 mg of PGE₁ -α-cyclodextrindissolved in 5 ml of water for injections were introduced into apresterilized 500 ml graduated flask and made up to 500 ml with theprepared emulsion, and a sample was taken for particle measurements. Thesolution containing active substance was transferred under asepticconditions and using a Dispensette with a capacity of 2 ml/single doseto a height of about 1 cm into presterilized vials. The charged vialswere then provided with stoppers, placed in the lyophilizer and frozenat -45° C. for 5 hours. The subsequent lyophilization process wascarried out as shown in the following table.

    ______________________________________             Initial       Final    Time     temperature   temperature                                     Pressure    (hours)  (°C.)  (°C.)                                     (μbar)    ______________________________________    30       -40           -40       100    10       ˜40     25        100    15        25           25         1    ______________________________________

Subsequently the vacuum was removed with simultaneous introduction ofnitrogen, the vials were closed by hydraulic lowering of the stoppersand were removed from the lyophilizer under aseptic conditions after ithad been opened.

They contained cakes which appeared dry and homogeneous and whichspontaneously disintegrated on addition of water and formed an emulsion.

EXAMPLE 2

1.5 g of citric acid monohydrate, 2.34 g of trisodium citrate dihydrateand 60 g of lactose were dissolved by heating in 476 g of water forinjections. In addition, 12.0 g of glycerol polyethylene glycolricinoleate and 18.0 g of diacetylated monoglycerides with a hydroxylnumber of 4 were dissolved in 30 g of absolute ethanol with gentleheating and under a nitrogen atmosphere. Subsequently, in accordancewith the preparation process described in Example 1, the emulsion wasprepared, the active substance was incorporated, samples were taken forparticle measurement, and lyophilization was carried out.

Likewise as in Example 1, the product cakes which have been formedappeared dry and homogeneous and spontaneously disintegrated to anemulsion on addition of water.

Particle size distribution

To examine the effect of lyophilization and redispersion on the particlesize, the volume distributions of the emulsion particles beforelyophilization and after freeze-drying and redispersion with water hadtaken placed were determined by laser light scattering (Malvern MasterSizer, Series 3.01, Malvern Instruments Limited, Spring Lane South,Malvern, Worcestershire, WR14 1AQ, UK). Comparison of the volumedistributions reveals even the slightest changes in the particularlycritical larger particles because the latter by their nature contributea larger portion of the total volume. The following table shows theresults of measurement of the particle size determination of emulsionsprepared according to the invention as in the examples. They contain themaximum particle sizes which characterize the volume distributions andwhich, together with the smaller particles lying below them in eachcase, include 10%, 50%, 90% or 99% of the total volume.

    ______________________________________               Maximum size of the particles               which, together with the smaller               particles in each case, comprise               the following volume proportions               10%   50%       90%     99%    ______________________________________    Example 1    before lyophilization                 0.20 μm                         0.39 μm                                   0.98 μm                                         5.07 μm    after lyophilization                 0.19 μm                         0.35 μm                                   1.02 μm                                         3.32 μm    and redispersion with    water    Example 2    before lyophilization                 0.19 μm                         0.38 μm                                   1.09 μm                                         4.56 μm    after lyophilization                 0.20 μm                         0.40 μm                                   1.17 μm                                         4.56 μm    and redispersion with    water    ______________________________________

It is clear that the particle size distributions of the emulsionsreconstituted by addition of water agree with the particle sizedistributions of the initial emulsions.

We claim:
 1. An anhydrous emulsion composition comprising at least oneactive substance, and at least one cryoprotection/bulking agent, whereinafter reconstitution with water affords a water-containing emulsion withsubstantially identical particle size distribution as the originalwater-containing emulsion.
 2. The composition of claim 1 which furthercomprises hydrophilic emulsifiers and acetylated monoglycerides.
 3. Thecomposition of claim 2 wherein the hydrophilic emulsifier comprisesethoxylated triglycerides or polyoxyethylene hydroxy fatty acid estersand the acetylated monoglycerides contain unsaturated double bonds. 4.The composition of claim 2 wherein the hydrophilic emulsifier isglycerol polyethylene glycol ricinoleate or polyoxyethylene 66012-hydroxystearate and the acetylated monoglycerides are diacetylatedmonoglycerides or a mixture of diacetylated and partially acetylatedmonoglycerides.
 5. The composition of claim 2 comprising about 2 to 40wt % acetylated monoglycerides.
 6. The composition of claim 5, whereinthe acetylated monoglycerides comprise about 20 wt % of partiallyacetylated monoglycerides.
 7. The composition of claim 2 wherein thehydrophilic emulsifiers and the acetylated monoglycerides are present ina weight ratio of about 1:10 to 2:1.
 8. The composition of claim 7wherein said weight ratio is about 2:3.
 9. The composition of claim 1wherein the active substance is a prostaglandin.
 10. The composition ofclaim 9 wherein said prostaglandin is PGE₁.
 11. The composition of claim1 wherein said cryoprotection/bulking agent comprises physiologicaltolerated mono-, di- or oligosaccharides.
 12. The composition of claim11 wherein said saccharides are selected from lactose, sorbitol, andmannitol.
 13. The composition of claim 1 which further comprises anantioxidant.
 14. The composition of claim 13 wherein said antioxidant isα-, β-, Γ-, or δ-tocopherol or a physiologically tolerated buffer saltor mixture thereof.
 15. The composition of claim 14 wherein thephysiologically tolerated salt is a phosphate, succinate or acetate. 16.The composition of claim 14 wherein the antioxidant is α-tocopherol. 17.The composition of claim 1 wherein said composition, afterreconstitution with water, has an average particle diameter of about 0.1μm to about 3 μm.
 18. The composition of claim 17 wherein the averageparticle diameter is about 0.2 μm to about 1.0 μm.